TW201920677A - Compositions, methods and uses for dengue virus serotype-4 constructs - Google Patents

Abstract

Embodiments herein report compositions, methods and uses for dengue-4 (DEN-4) virus constructs. Some embodiments concern a composition that includes, but is not limited to, DEN-4 virus constructs disclosed herein that alone or in combination with other constructs can be used in a vaccine composition to induce an immune response in a subject. In certain embodiments, compositions can include constructs of more than one serotypes of dengue virus, such as dengue-1 (DEN-1) virus, dengue-2 (DEN-2) virus, or dengue-3 (DEN-3) virus in combination with DEN-4 virus constructs disclosed herein. In other embodiments, DEN-4 constructs disclosed herein can be combined in a composition with other flavivirus constructs to generate a vaccine against more than one flavivirus. Other embodiments provide methods and uses for DEN-4 virus constructs in vaccine compositions that when administered to a subject induce an immune response in the subject against DEN-4 that is improved by modified constructs compared to other vaccine compositions.

Description

   Composition, method and application of dengue virus serotype 4 construct        Federally funded research    

Some specific examples disclosed here are sponsored in part by the National Institutes of Health fund number R43 AI084291-01. The U.S. government has certain rights to implement the invention.

    Field of invention    

Specific examples here describe the composition, method, and use of a dengue 4 (DEN-4) virus construct. Some specific examples relate to a composition including, but not limited to, a DEN-4 virus construct, alone or in combination with other agents, which can be used in a vaccine composition. In certain embodiments, the composition may include chimeric constructs of more than one dengue virus serotype, such as dengue 1 (DEN-1) virus, dengue 2 (DEN-2) virus, or dengue 3 (DEN- 3) Viruses, in combination with DEN-4 virus chimera constructs, in the form of two-, three- or four-valent formulations. In other specific examples, the DEN-4 chimera construct (dengue-dengue chimera) disclosed herein can be combined with other flavivirus constructs. Some specific examples include DEN-4 chimeric constructs with components (such as structural elements) of other dengue serotypes. Other specific examples provide a method and use of a DEN-4 virus chimera construct in a vaccine composition. When administered to an individual, it induces an immune response against DEN-4 in the individual, which is similar to other constructs. Than, has been improved.

    Background of the invention    

Infection with dengue virus can cause painful fevers of various severity. To date, four dengue serotypes have been identified: dengue 1 (DEN-1), dengue 2 (DEN-2) or dengue 3 (DEN-3), and dengue 4 (DEN-4). Dengue fever is caused by infection with the dengue virus. Dengue virus serotypes 1-4 can also cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The most serious infections, DHF and DSS, can be life threatening. Dengue virus causes 5-10 million cases of debilitating dengue fever, 500,000 cases of DHF / DSS and more than 20,000 deaths each year. To date, no effective vaccine has provided protection against dengue fever, and there is no medication for this disease. Efforts to control mosquitoes have been ineffective in preventing outbreaks of dengue fever in endemic areas, or in preventing further geographical spread of the disease. An estimated 3.5 billion people are at risk from dengue virus infection. In addition, dengue virus is the main cause of fever in travelers to endemic areas such as Asia, Central and South America, and the Caribbean.

All four dengue virus serotypes are endemic in tropical regions of the world and pose the most serious mosquito-borne virus threat to humans in tropical regions of the world. Dengue virus is mainly transmitted to humans by Aedes aegypti. Infected by one serotype of dengue virus, will not be repeatedly infected by this serotype throughout life, but it cannot prevent secondary infection of one of the other three dengue virus serotypes. In fact, a previous infection with a dengue virus serotype and a subsequent secondary infection with a different serotype increased the risk of serious illness (DHF / DSS). The development of effective vaccines is an important way to prevent and control this worldwide raging disease.

    Summary of invention    

Specific examples here relate to the composition, method, and use of the DEN-4 virus chimera construct. In some specific examples, the composition may include a DEN-4 virus chimera construct, alone or in combination with other dengue virus serotype constructs, or live attenuated dengue virus of the same or other serotype, or other capable of targeting the target virus ( (Eg, dengue virus) flavivirus constructs that elicit an immune response. Other specific examples may include a composition having a live attenuated virus construct against DEN-4, and optionally one or more live attenuated virus constructs against DEN-1, DEN-2, and DEN-3. In other specific examples, an immunogenic composition is provided, which includes a DEN-4 live attenuated chimeric virus construct that increases immunogenicity when introduced into a body. According to these specific examples, these live attenuated virus constructs can be used alone or in combination with one or more other DEN-1, DEN-2, and DEN-3 constructs, as well as pharmaceutically acceptable excipients to produce anti-dengue fever Viral serotype vaccine formulation. In certain embodiments, a monovalent, bivalent, trivalent, or tetravalent pharmaceutically effective formulation is produced against one or more dengue viruses. In some specific examples, the immunogenic composition may include one or more of DEN-1, DEN-2, and DEN-3 dengue-dengue chimeric constructs, and the chimeric DEN-4 constructs disclosed herein Thing.

In some specific examples, including the DEN-4 construct of the present invention, in combination with one or more of DEN-1, DEN-2, and DEN-3, the immunogenic composition can be used when a single vaccine is administered, Provides simultaneous protection against two or more dengue virus serotypes. In other specific examples, an immunogenic composition comprising DEN-1, DEN-2, DEN-3, and modified or mutated DEN-4 constructs of the specific examples disclosed herein can be administered in a single body, It is used to induce an improved immunogenic response against each dengue virus serotype and to reduce interference with the immune response to DEN-4.

In some specific examples, the DEN-4 construct may include a dengue-dengue chimeric construct with an adaptive mutation in the structural or non-structural region of DEN-4. In other embodiments, the DEN-4 construct may include another dengue virus serotype, the backbone of DEN-1, DEN-2, or DEN-3. In yet other specific examples, the chimeric construct may include a DEN-2 backbone, where the structural or non-structural region of DEN-4 replaces the structural or non-structural region of DEN-2. According to these specific examples, the DEN-2 backbone can include any live attenuated DEN-2 virus. In other specific examples, the DEN-2 backbone may include a live attenuated DEN-2 PDK-53 virus as a backbone, where the live attenuated DEN-2 PDK virus further includes a prM (pro-membranous) having DEN-4 and (Envelope) One or more of the structural proteins. In addition, in order to improve the individual's immune response to DEN-4 at the time of administration, the DEN-2 PDK-53 backbone may include additional mutations or mutant responses to DEN-2 PDK-53.

In some specific examples, in order to improve the replication efficiency of both the virus used for production in a test tube and in vivo as a construct that elicits an immune response to DEN-4, the strain currently known as DENVax-4 was modified (Sequence identification number: 21) The dengue chimeric construct made the shell / PrM linker containing the DEN-2 backbone so as to be more genetically similar to DEN-4 than DEN-2. The current DENVax-4 strain has a capsid / PrM sequence that is consistent with DEN-2 instead of DEN-4, and will produce a different RNA secondary structure than DENV-4.

In some specific examples, the structural protein gene may include the prM and E genes of DEN-4 on another dengue virus backbone to make a dengue-dengue chimera. For example, in some specific examples, DEN-4 constructs may include the so-called DENVax-4e (shell 107 cysteine to tyrosine; DenVax-4b backbone, modified at the shell / prM connector ), DENVax-4f or DENVax-4h (the mantle 417 Glu becomes Lys) (see, for example, Figure 4). Here, for certain constructs, the DEN-2 PDK-53 backbone has one or more types of wild type DEN-2 responses (eg, in non-coding regions (NCR) or non-structural regions (NS1, etc.)) and one or more mutations in DEN-4 structural regions (eg, prM or E), encoding one or more Structural protein of DEN-4 (e.g., strain 1036). The modified DEN-4 constructs disclosed herein may include a modified attenuated DEN-2 PDK-53 backbone, with one or more modified structural proteins of the DEN-4 strain 1036. In some specific examples, the presence of one or more mutations in the live attenuated DEN-2 PDK-53 virus can be reverted to a control amino acid or another amino acid to generate a structure herein, which can be used in Does not affect its attenuating effect, but can affect the growth and / or replication of DEN-4 virus, resulting in a modified DEN-2 / DENV-4 construct with increased immunogenicity. In some specific cases, this recovery effect can increase growth and / or replication.

In other specific examples, in order to produce an improved immune response while maintaining safety and virus attenuation, the modified DEN-4 construct can be incorporated into one or more of the structural regions and / or non-structural aspects of DEN-4 Zone mutation. For example, DEN-2 / DEN-4 modified or mutated dengue-dengue chimeras may contain mutations in one or more non-structural regions of the DEN-2 PDK-53 backbone, such as in NS2A and NS4A, and / Or 5 'non-coding region (5'NCR). In another specific example, the modified DEN-4 chimera construct may include NS2A of DEN-2 16681 by reverting mutations at NS2A and NS4A at PDK-53 (eg, ML substitution at NS4A). And NS4A. Some specific examples include a modified DEN-4 chimera construct having 5'NCR, DEN-2 16681-5'NCR, NS2A, and DEN-2 16681 by corresponding mutations in the DEN-2 PDK-53 backbone of the target construct. NS4A. Other specific examples may include a modified DEN-4 chimera construct having 5'NCR of DEN-2 16681 by reverting to a corresponding mutation in the DEN-2 PDK-53 backbone. The modified DEN-4 chimera construct may also include a DEN-2 PDK-53 backbone and one or more structural proteins encoding the DEN-4 strain H241. Any DEN-4 construct protein can be considered to replace the structural or non-structural regions of dengue serotype 2 (eg, PDK-53 or modified PDK-53). In some specific examples, the modified DEN-4 construct contains live attenuated DEN-2 PDK-53 as a backbone, and a DEN-4 structural protein, and mutations can be introduced here to modify DEN-4 (eg, strain 1036 ) Structure area.

In other specific examples, mutations can be introduced into the DEN-4 shell / prM linker amino acid sequence in order to increase the immunogenicity of the construct containing the mutation. For example, the mutation in DEN-4 may be a Cys-Tyr mutation at the capsid position 107 of DEN-4. In other specific examples, mutation of the cysteine at position 107 to any other aromatic amino acid having a hydrophobic fluorene chain can be considered (see, for example, DEN-4e). Other DEN-2 PDK-53 responses to the chimeric constructs can be found in NS2A or NS4A. Still other specific examples include the DEN-4 construct, where the DEN-2 backbone contains PDK-53 (MVS, sequence identification number: 21), and the amino acid position 102-107 of the shell region of PDK-53 is converted. The corresponding amino acid of DEN-4 produces DENV-4b (see, for example, Figure 4). These skeletal constructs may then further comprise cysteine in the shell region, which becomes an aromatic amino acid (eg, tyrosine, tryptophan, etc.) in situ. In some specific examples, the structure is represented by a sequence identification number: 22 or a sequence identification number: 23.

Other DEN-4 constructs disclosed herein may include amino acid substitutions at the mantle position 417 of the DEN-4 strain, for example, the sequence of the 1036 strain or an equivalent strain position thereof, provided herein with DEN DK-53 backbone of dengue-2 structural protein-4 (MVS DEN2 / 4, sequence identification number: 21). Specific examples include a further mutated mantle position 417, which changes from a negatively charged to a positively charged side chain amino acid (eg, lysine). It is expected that any positively charged side chain will provide increased immunity to the DEN-4 construct without affecting its safety or attenuating effect. In some specific examples, the structure is represented by a sequence identification number: 24 or a sequence identification number: 25.

In some specific examples, the DEN-2 PDK-53 response of the DEN-4 chimeric construct has 5'NC, NS1, and NS3 mutations that can be found in the DEN-2 PDK-53 MVS, with other responses or mutation. It has been demonstrated that these three mutations may be important for attenuating effects such as smaller plaque size, reduced growth rate, lower titer, increased temperature sensitivity, and reduced neurotoxicity compared to the control group.

In other specific examples, the DEN-2 PDK-53 genomic backbone can be used to generate chimeric constructs of DEN-1 and DEN-3. Here, one or more structural protein genes of the DEN-2 PDK-53 genome can be determined by DEN. One or more structural protein gene substitutions of -1 and DEN-3. These constructs can be in a single chimera with a DEN-2 PDK-53 backbone, including a combination of both DEN-1 and DEN-3. In some specific examples, the structural protein may be the C, prM or E protein of DEN-1 and / or DEN-3. In some specific examples, the structural protein genes include prM and E genes of DEN-1 or DEN-3. These hybrid / chimeric viruses express surface antigens of DEN-1, DEN-3, or DEN-4, while retaining the attenuated phenotype of the parent DEN-2. In some specific examples, these structures are represented by sequence identification number: 15, DEN-2 / DEN-1 and sequence identification number: 19, DEN-2 / DEN-3. Here, these structures can be used for Two-, three-, or four-valent compositions disclosed herein.

In some specific examples, the structures disclosed herein may include chimeric structures of DEN-4, DEN-2, DEN-1, and DEN-3, which can express DEN-1, DEN-3, and DEN-4. Surface antigen, and the use of attenuated DEN-2 PDK-53 virus as a backbone.

Some specific examples disclose methods for making modified or mutated DEN-4 constructs and applying them to any vaccine composition, including, but not limited to, a single vaccine composition having only a DEN-4 construct; Hybrid single vaccine composition of immune response to one or more dengue virus serotypes; with chimeric (and non-chimeric) constructs disclosed herein in combination with other capable of inducing flavivirus and one or more dengue fevers including DEN-4 Viral serotype immune response hybrid flavivirus constructs mixed single vaccine composition.

The following drawings form a part of this specification and are used to further illustrate some specific examples. Some specific examples can be better understood by referring to one or more of these drawings alone or in combination with the detailed description of the specific specific examples presented.

Figures 1A-1B illustrate (A) representative schematic diagrams of the structure of DEN-2 PDK-53 and non-structural genes, and (B) certain chimeric dengue virus constructs of some specific examples disclosed herein, respectively, in Demonstration results of structural proteins expressing different dengue virus serotypes in the DEN-2 PDK-53 backbone.

FIG. 2 is a table illustrating exemplary experimental designs for the analysis of certain vaccines including the DEN-4 chimeric constructs disclosed herein in non-human primates.

Figure 3 depicts an experimental demonstration of the yield of DEN-4 neutralizing antibody titers produced from animal models using vaccine compositions with certain specific examples of DEN-4 chimeric virus constructs disclosed herein .

Figure 4 depicts a schematic diagram of certain DEN-4 chimeric constructs disclosed in some specific examples herein, alone or in combination with other live attenuated virus constructs, for use in vaccine formulations.

FIG. 5 is a table illustrating plaque parameters of some DEN-4 virus constructs and some exemplary data of Vero cell titer and cell growth analysis for some specific examples disclosed herein.

Figures 6A-6F are photographs of exemplary ImmunoFoci results that provide demonstration experiments using certain DEN-4 chimera constructs, including DEN-4 wild type (A), DENVax-4 (B), DENVax-4e (C), DENVax-4h (D), DENVax-4i (E), and DENVax-4j (F).

Figure 7 is a graph depicting the viral titers of various DEN-4 chimeric constructs in mammalian cells over time, referred to as "days after infection".

FIG. 8 is a graph showing changes in the virus titers of DEN-4 chimeric constructs in mosquito cells over time, referred to as "days after infection".

FIG. 9 is a flowchart describing an acceptable animal model for testing various DEN-4 chimeric constructs to induce an immune response to DEN-4 in an animal model.

Figure 10 illustrates the alignment of various DEN-4 chimeric constructs, illustrates common sequences, and exemplary mutations and / or reversions.

Figure 11 illustrates an alignment of various DEN-4 chimeric constructs, illustrates common sequences, and exemplary mutations and / or reversions.

Figure 12 illustrates a schematic diagram describing a live attenuated virus construct of the DEN-2 virus.

Figure 13 illustrates an exemplary schematic diagram of the selection of various DEN-4 constructs disclosed herein.

Figure 14 depicts a graph comparing the growth of various DEN-4 constructs in cell lines.

Figure 15 depicts a graph comparing the growth of various DEN-4 constructs in mosquito cells.

Figure 16 depicts an exemplary histogram comparing the immunogenicity of various DEN-4 constructs disclosed herein in mice.

Figure 17 depicts an exemplary diagram (expressed as survival rate) illustrating the use of various DEN-4 constructs in mice to protect and immunize against the challenge of dengue.

Figure 18 depicts a schematic diagram of a DEN-4 construct considered for use in this formulation.

Figure 19 depicts a schematic diagram of a DEN-4 construct considered for use in this formulation.

Figure 20 depicts a schematic illustration of various DEN-4 constructs considered for use herein.

Figure 21 depicts the levels of various dengue constructs after mice were vaccinated, expressed as days after vaccination.

Figure 22 depicts the level of various dengue constructs after vaccination in mice, expressed as days after vaccination.

Figure 23 depicts the levels of various dengue constructs after vaccination in mice, expressed as days after vaccination.

Figure 24 depicts the level of various dengue constructs after vaccination in mice, expressed as days after vaccination.

    Definition    

As used herein, "a" means one or more than one item.

The term "individual" used herein may include, but is not limited to, mammals such as humans (e.g., adults and adolescents), or domestic or wild mammals, such as dogs, cats, other domestic pets (e.g., hamsters, Guinea pig, mouse, rat), ferret, rabbit, pig, horse, cow, marmot, wild rodent or zoo animal.

The terms "chimeric construct", "viral chimera", "chimeric virus", "flavivirus chimera" and "chimeric flavivirus" as used herein mean a nucleoside comprising a portion of the dengue-2 virus Acid sequence, and additionally contains a construct that is not derived from the dengue-2 virus or from a different dengue virus serotype or a different flavivirus nucleotide sequence. A "dengue chimera" contains at least two different dengue virus serotypes. Examples of other dengue viruses or flaviviruses include, but are not limited to, from dengue-1 virus, dengue-3 virus, dengue-4 virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, Sequences of tick-borne encephalitis virus, yellow fever virus, and any combination thereof.

As used herein, "nucleic acid chimera" means a construct disclosed herein that includes a nucleic acid sequence that includes a portion of the nucleotide sequence of a dengue-2 virus and one or more nucleotide sequences that are related to the dengue-2 virus Nucleotide sequences from different sources. In contrast, any chimeric flavivirus, any dengue chimera, or flavivirus chimera disclosed herein can be considered as examples of nucleic acid chimeras.

    Description    

In order to make each specific example more detailed, various exemplary compositions and methods will be described in the following paragraphs. For those familiar with the art, it is clear that the implementation of each specific example does not require the use of all or even some of the specific details outlined here, but that the concentration, number of times, and other specific details can be modified through routine experimentation. In some cases, well-known methods or components are not included in the description.

According to specific examples of the present invention, it is possible to use meristem, protein chemistry, microorganisms, and recombinant DNA techniques commonly used in the art. These techniques are explained fully in the literature. See, eg, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y .; Animal Cell Culture, R.I. Freshney, ed., 1986).

Specific examples herein are compositions, methods, and uses for inducing an improved immune response against DEN-4 in an individual, alone or in combination with one or more agents for inducing anti-dengue virus serotypes or flaviviruses. According to these specific examples, live attenuated dengue virus and nucleic acid chimeras of DEN-4 are produced and used in the immunogenic composition disclosed herein. Some specific examples relate to modified or mutated DEN-4 constructs. Some specific examples relate to introducing mutations and / or reversions into a DEN-4 chimeric construct to modify the amino acid sequence of the chimeric construct. In some specific examples, introducing a DEN-4 chimeric construct in order to modify the mutation and / or reversion of the amino acid sequence of the chimeric construct may include the use of mutagenesis techniques including, but not limited to, A mutation upstream of the C / prM break site of the chimeric construct (referred to as DENVax-4 (sequence identification number: 21)) is known.

Specific examples here relate to the composition, method, and use of the DEN-4 virus chimera construct. In some specific examples, the composition can include a DEN-4 virus chimera construct alone, or in combination with other dengue virus serotype constructs, or can induce an immune response to a target virus (eg, dengue virus or other flavivirus) Live attenuated dengue virus or other flavivirus constructs of the same or other serotypes. Other specific examples may include live attenuated virus constructs against DEN-4 and optionally one or more live attenuated virus constructs against DEN-1, DEN-2, and DEN-3. In still other specific examples, an immunogenic composition is provided that includes a DEN-4 live attenuated chimeric virus construct that has increased immunogenicity when introduced into a body compared to other known constructs Sex. According to these specific examples, these live attenuated virus chimera constructs can be used alone or in combination with one or more other DEN-1, DEN-2, and DEN-3 constructs (eg, live attenuated virus or chimera) And a pharmaceutically acceptable excipient to produce a vaccine formulation against one or more dengue virus serotypes. In certain embodiments, a monovalent, bivalent, trivalent, or tetravalent pharmaceutically effective formulation is produced against one or more dengue viruses. In certain embodiments, the immunogenic composition may include one or more of DEN-1, DEN-2, and DEN-3 dengue-dengue chimeric constructs, or a live attenuated dengue virus, as disclosed herein Chimeric DEN-4 construct.

In some specific examples, the DEN-4 construct of the present invention and the immunogenic composition of one or more of DEN-1, DEN-2 and DEN-3 can be used for administration as a single vaccine to provide anti- Simultaneous protection of two or more dengue virus serotypes. In other specific examples, an immunogenic composition comprising DEN-1, DEN-2, DEN-3 and the modified or mutated DEN-4 construct of the present invention disclosed herein can be administered in a body. It is used to induce immune responses against various dengue virus serotypes, and the interference with the immune response to DEN-4 is reduced here.

In some specific examples, the DEN-4 construct may include a dengue-dengue chimeric construct having an adaptive mutation in the structural or non-structural region of DEN-4. In other specific examples, the DEN-4 construct may include the backbone of another dengue virus serotype DEN-1, DEN-2, or DEN-3. In yet other specific examples, the chimeric construct may include a DEN-2 backbone, where the structural or non-structural region of DEN-4 replaces the structural and / or non-structural region of DEN-2. According to these specific examples, the DEN-2 backbone can include any live attenuated DEN-2 virus that is safe and effective and can induce an immune response to DEN-2. In other specific examples, the DEN-2 skeleton can include live attenuated DEN-2 PDK-53 (replicated 53 times in primary dog kidney cells (PDK)) or derived from the DEN-16681 strain as a skeleton. The attenuated DEN-2 PDK-53 virus further includes one or more of the structural proteins of prM (promoter) and E (envelope) structural proteins of DEN-4. In addition, in order to enhance the immune response to DEN-4 during administration, the DEN-2 PDK-53 backbone may include additional mutations or reversions of DEN-2 PDK-53. (See, for example, Figure 4).

In some specific examples, the structural protein gene may include the prM and E genes of DEN-4 on another dengue virus backbone to make a dengue-dengue chimera. For example, in certain embodiments, DEN-4 constructs may include such constructs as DENVax-4e, DENVax-4f, or DENVax-4h (see, for example, Figure 4), where the DEN-2 PDK-53 backbone With one or more responses to wild-type DEN-2 amino acids (e.g., in the non-structural region (NCR) or non-structural region (NS1, etc.)) and one or more mutations in the DEN-4 structural region (e.g., prM or E), and also encode one or more structural proteins of DEN-4 (eg, strain 1036). The modified DEN-4 constructs disclosed herein may include a modified attenuated DEN-2 PDK-53 backbone with one or more modified structural regions of the DEN-4 strain 1036. In some specific examples, one or more mutations present in the live attenuated DEN-2 PDK-53 virus can be reverted to a control amino acid or another amino acid to produce a construct herein that will modify The DEN-2 / DENV-4 chimeric construct (when compared with the MVS sequence, sequence identification number: 21), does not affect its attenuating effect, but can affect the growth and / or replication of the DEN-4 virus In this case, increase the immunogenicity. In some specific cases, this recovery may result in increased growth and / or replication.

In other specific examples, the modified DEN-4 construct may incorporate mutations in one or more of the structural and / or non-structural regions of DEN-4 in order to generate an improved immune response while maintaining safety and reducing Toxic constructs. For example, DEN-2 / DEN-4 modified or mutated dengue-dengue chimeras may contain mutations at one or more non-structural regions of the DEN-2 PDK-53 backbone, such as NS2A and NS4A, and And / or a mutation at a 5 'non-coding region (5'NCR). In another specific example, the modified DEN-4 chimera construct may include NS2A of DEN-2 16681 by reverting mutations at NS2A and NS4A at PDK-53 (eg, ML substitution at NS4A). And NS4A. Some specific examples include modified DEN-4 chimera constructs with 5 ' NCR, NS2A, and NS4A of DEN-2 16681 by corresponding mutations in the DEN-2 PDK-53 backbone of the target construct. Other specific examples may include a modified DEN-4 chimera construct having 5'NCR of DEN-2 16681 by reverting a corresponding mutation in the DEN-2 PDK-53 backbone. The modified DEN-4 chimera construct may also include a DEN-2 PDK-53 backbone and one or more structural proteins encoding the DEN-4 strain H241. Any structural protein of DEN-4 can be considered to replace the structural or non-structural regions of dengue serotype 2 (eg, PDK-53 or modified PDK-53). In some specific examples, the modified DEN-4 construct contains live attenuated DEN-2 PDK-53 as a backbone, and a DEN-4 structural protein, and mutations can be introduced here to modify DEN-4 (eg, a strain 1036). In some specific examples, mutations can be introduced into the DEN-4 shell / prM linker amino acid sequence in order to increase the immunogenicity of the construct containing the mutation. For example, the mutation in DEN-4 may be a C-Y mutation at the capsid position 107 of DEN-4 (see, for example, DENVax-4e). According to these specific examples, on the modified PDK-53 backbone (DENV-4b), cysteine can be mutated to an aromatic amino acid (eg, tyrosine or others). Other mutations can include amino acid substitutions at mantle position 417 (Phenylamine, E) in the DEN-4 1036 strain sequence (see, for example, DENVax-4h) or at an equivalent position in another DEN-4 Here, the negatively charged amino acid is replaced by a positively charged amino acid (eg, lysine, arginine, histidine, etc.) with a charged side chain. Other DEN-2 PDK-53 responses to the chimeric construct can be found in the NS2A or NS4A regions.

In other specific examples, the DEN-2 PDK-53 genomic backbone can be used to generate chimeric dengue virus constructs of DEN-1 and DEN-3, where one or more of the DEN-2 PDK-53 genome is structural or non-structural The protein gene can be replaced by one or more structural proteins or non-structural genes of DEN-1 and DEN-3. These constructs can be in a single chimera with a DEN-2 PDK-53 backbone, including a combination of both DEN-1 and DEN-3 structural or non-structural genes. In some specific examples, the structural protein may be the C, prM or E portion of DEN-1 and / or DEN-3. In some specific examples, the structural protein genes include prM and E genes of DEN-1 or DEN-3, or a combination thereof. These hybrid / chimeric viruses can express surface antigens of DEN-1, DEN-3 or DEN-4, while retaining the attenuated phenotype of the parent DEN-2.

In some specific examples, the structures disclosed herein may include chimeric structures of DEN-4, DEN-2, DEN-1, and DEN-3, which can express DEN-1, DEN-3, and DEN-4. Surface antigen, and use attenuated DEN-2 PDK-53 or live attenuated DEN-2 16681 virus as a backbone. In addition, constructs that are part of a pharmaceutical composition may include other agents such as other live attenuated viruses (eg, DEN-2, other flaviviruses). In addition, other agents used in these compositions may include other pharmaceutically acceptable antiviral agents, adjuvants or stabilizers to reduce degradation of live attenuated viruses.

Some specific examples herein disclose methods for making modified or mutated DEN-4 constructs and applying them to any vaccine composition against DEN-4, including, but not limited to, a single DEN-4 construct Vaccine compositions; mixtures of dengue virus constructs of a single vaccine composition capable of eliciting an immune response against two or more dengue virus serotypes; chimera (and non-chimerism) disclosed herein in a single vaccine composition Constructs in combination with other flavivirus constructs capable of inducing an immune response to different flaviviruses (eg, yellow fever, West Nile, Japanese encephalitis, etc.) and one or more dengue virus serotypes including DEN-4.

In other specific examples, other combinations may be considered for use with the DEN-4 constructs disclosed herein. For example, dengue virus serotype 1 wild-type virus was regenerated 13 times in PDK cells and named DEN-1 PDK-13 virus. Other vaccine candidates are DEN-2 PDK-53, DEN-3 PGMK-30 / FRhL-3 (eg, 30 passages in primary green monkey kidney cells and 3 passages in rhesus monkey fetal lung cells ) And DEN-4 PDK-48. These four candidate vaccine viruses were derived from tissue-cultured wild-type parents DEN-1 16007, DEN-2 16681, DEN-3 16562, and DEN-4 1036, respectively. Any of these existing live attenuated dengue viruses can be considered for use in combination with the DEN-4 chimeric virus constructs disclosed herein.

Previous human clinical trials of these attenuated viruses have indicated that DEN-2 PDK-53 has the lowest infectious dose in humans (5 plaque-forming units or 50% of the minimum infectious dose of PFU) and has a strong immunogenicity Without any significant security issues. Candidates for DEN-1 PDK-13, DEN-3 PGMK-30 / FRhL-3, and DEN-4 PDK-48 vaccine viruses have higher minimum infectious doses of 50%, 10,000, 3500, and 150 PFU in humans, respectively.

The DEN-2 PDK-53 virus vaccine candidate, hereinafter referred to as PDK-53, has many measurable biomarkers related to attenuation, including temperature sensitivity, small plaques, reduced replication in mosquito C6136 cell culture, Reduced replication in intact mosquitoes, loss of neurotoxicity to young mice, and reduced incidence of viremia in monkeys. Clinical trials of the candidate PDK-53 vaccine have proven its safety and immunogenicity in humans. Furthermore, PDK-53 vaccine induces dengue virus-specific T-cell memory responses in human vaccine recipients.

An immunogenic flavivirus chimera having a dengue-2 virus backbone and at least one structural protein of dengue-4 virus can be used to prepare a dengue virus chimera, and a method for generating the dengue virus or flavivirus chimera is described here. Provide the immunogenic flavivirus chimera, alone or in combination, in a pharmaceutically acceptable carrier to become an immunogenic composition for subjecting an individual to one or more dengue virus or flavivirus strains ( Infections such as dengue virus serotype DEN-4, alone or in combination with DEN-2, DEN-3 and DEN-1) are minimized, prevented or immune. When combined, the immunogenic flavivirus chimera can be used as a multivalent vaccine, providing simultaneous protection against more than one flavivirus strain. In certain embodiments, the flavivirus chimera is conjugated in an immunogenic composition and is used as a bivalent, trivalent, or tetravalent vaccine against a known dengue virus serotype, or by including a Nucleic acids from various flaviviruses provide immunity to other pathogenic flaviviruses. The nucleic acid sequences of each of DEN-1, DEN-2, DEN-3, and DEN-4 can be used to generate probes for the detection of dengue virus in biological samples, for example, to evaluate the effectiveness of vaccines and / or dengue The extent of the viral infection.

In some specific examples, the non-pathogenic, immunogenic chimeras provided herein contain a non-structural protein gene of an attenuated dengue-2 virus (eg, PDK-53) or an equivalent thereof, and one or more yellow The structural protein gene of the virus or its immunogenic protein portion (which can induce immunity against the flavivirus in an individual). For example, some specific examples relate to a chimera that has the attenuated dengue-2 virus PDK-53 genome as the viral backbone and one or more structural proteins encoding the shell, protrusive membrane / membrane or membrane sleeve of the PDK-53 genome. Genes, or a combination thereof, wherein the structural protein is replaced by one or more corresponding structural protein genes from DEN-4 virus or other flaviviruses to be combated, such as different flaviviruses or different dengue virus serotypes. According to these specific examples, the PDK-53 backbone is further mutated or reverted to increase the immunogenicity of the construct. Furthermore, the nucleic acid chimeras disclosed herein may have the functional properties of attenuated dengue-2 virus and are non-pathogenic, but express antigens of the structural gene products of DEN-4 in addition to other flaviviruses Epitope and is immunogenic (eg, elicits an immune response to an individual's gene product). Mutations and / or reversions do not affect the attenuating effect and / or safety of the chimeric construct.

In another specific example, the nucleic acid chimera may have, but is not limited to, a first nucleotide sequence encoding a non-structural protein derived from attenuated dengue-2 virus, and a structure derived from dengue-4 virus A nucleic acid chimera of the protein's second nucleotide sequence, alone or in combination with another flavivirus. In other specific examples, the attenuated dengue-2 virus may be strain PDK-53 or 16681. Some specific examples include structural proteins of one or more of C, prM or E of dengue or other flaviviruses. Examples of flaviviruses from which structural proteins can be selected include, but are not limited to, DEN-1, DEN-2, DEN-3, West Nile virus, Japanese encephalitis virus, Saint Louis encephalitis virus, yellow fever virus, and ticks Metoencephalitis virus, and the DEN-4 construct disclosed herein. In other specific examples, the structural protein may be selected from non-flavivirus species that are very similar to flaviviruses, such as hepatitis C virus.

Other aspects disclosed herein include: chimeric viruses may include nucleotides and amino acid substitutions, deletions or insertions in, for example, DEN-2 PDK-53; such changes may reduce the effect on DEN-4 virus Disturbance of the immune response. These modifications can be made in structural as well as non-structural proteins, alone or in combination with the modification examples disclosed herein.

Specific examples herein include the structural and non-structural proteins of flaviviruses, which can be any protein including the following or any gene encoding the following: an intact protein, an epitope of the protein, or any containing, for example, five or Sequence of more fragments of amino acid residues.

Some specific examples disclosed herein provide a method for producing a chimeric virus of the present invention by using recombinant technology to insert a desired substitution into an appropriate backbone genome.

    Flavivirus chimera    

Dengue virus types 1-4 (DEN-1 to DEN-4) are mosquito-borne flavivirus pathogens. The flavivirus genome contains a 5'-noncoding region (5'-NC), followed by a capsid protein (C) coding region, followed by a proteomic membrane / membrane protein (prM) coding region, and then a envelope protein (E) coding region This is followed by a region encoding a non-structural protein (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5), and finally a 3 'non-coding region (3'NC) (see, for example, Figure 1A). The structural proteins of the virus are C, prM, and E, and the non-structural proteins are NS1-NS5. Structural and non-structural proteins are translated into a single polyprotein and then treated with cells and viral proteases.

    Structure of the dengue virus genome    

The flavivirus chimera construct can be formed by fusion and protein genes of nonstructural protein genes derived from one type or serotype of dengue virus, or viruses of the flaviviridae family, such as from different dengue viruses Type or serotype, or a structural protein gene from a virus species of the Flaviviridae family. Alternatively, the formation of the flavivirus chimeras disclosed herein is achieved by fusion of a non-structural protein gene from one of the dengue virus types or serotypes or a virus species of the Flaviviridae family. In addition, it will guide the synthesis of nucleotide sequences selected from the polypeptides or proteins of other dengue virus serotypes or other flaviviridae viruses.

In other specific examples, the non-pathogenic and immunogenic flavivirus chimeras provided herein contain a non-structural protein gene of attenuated dengue-2 virus or an equivalent thereof, and a flavivirus to which immunity is desired One or more of the structural protein genes, or an antigenic portion thereof.

Other flaviviruses suitable for the construction of flavivirus chimeras can be wild-type, toxic DEN-1 16007, DEN-2 16681, DEN-3 16562 and DEN-4 1036, and attenuated vaccine strains DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PMK-30 / FRhL-3, and DEN-4 PDK-48. Genetic differences between DEN-1, DEN-2, DEN-3, and DEN-4 wild-type / attenuated virus pairs and changes in the amino acid sequence encoded by the viral genome can be considered together. Any of the DEN-4 strains used herein contain mutations synonymous with the constructs considered and / or disclosed herein.

The sequence listing of DEN-2 PDK-53 is equivalent to the DEN-2 PDK-53-V mutant, in which the genomic nucleotide position 5270 is mutated from A to T, and the amino acid position of the polyprotein is 1725 or the amino group of the NS3 protein Acid position 250, containing valine residues. The DEN-2 PDK-53 variant without this nucleotide mutation, DEN-2 PDK-53-E, differs from PDK-53-V only in this position. DEN-2 PDK-53-E has an A at nucleotide position 5270, and amidine at polyprotein amino acid position 1725 and NS3 protein amino acid position 250. Of course, the specific examples herein may include modified DEN-2 PDK-53, which includes a back / mutation of one or more of these positions into a naturally derived sequence.

The sequence list of DEN-3 16562 is equivalent to the mutant strain in which the nucleotide position 1521 of the genome is T, and the amino acid position 476 of the polyprotein or the amino acid position 196 of the E protein contains leucine. The second variant, which appears in the DEN-3 16562 culture, has a T at nucleotide position 1521 and contains serine at amino acid position 476 of the polyprotein or amino acid position 196 of the E protein.

The sequence list of DEN-4 PDK-48 is equivalent to the genomic nucleotide position: 6957 is T, while the amino acid position of polyprotein 2286 and the amino acid position 44 of NS4B protein are phenylalanine, 7546 is T, and The amino acid position 2366 of the polyprotein and the amino acid position 240 of the NS4B protein are valine and 7623 is T. The amino acid position 2508 of the polyprotein and the amino acid position 21 of the NS5 protein are tyrosine. Mutant strain.

In some specific examples, the nomenclature is based on the DEN-2 virus specifically infecting the cloned plant skeleton and other flavivirus structural gene (prM-E or C-prM-E) inserts. DEN-2 is the dengue-2 backbone, followed by a strain with a structural gene inserted. Specific skeletal variants are reflected below. A special DEN-2 backbone variant constructing the chimera is indicated by a letter after the hyphen, parent 16681 (P), PDK-53-E (E) or PDK-53-V (V); The last letter indicates the C-prM-E structural gene from the parent (P) strain or its vaccine derivative (V), or the parent (P) or its vaccine derivative (V1) prM-E structural gene. For example, DEN-2 / 1-VP provides a chimera containing an attenuated DEN-2 PDK-53V backbone, which contains valine in NS3-250 and a C-prM-E gene from wild-type DEN-1 16007. DEN-2 / 1-VV provides the DEN-2 PDK-53V backbone and vaccine strains of dengue-1 and DEN-1 PDK-13; DEN-2 / 1-VP1 provides the DEN-2 PDK-53V backbone and PrM-E gene from type DEN-1 16007; DEN-2 / 3-VP1 provides the DEN-2 PDK-53V backbone and prM-E gene from wild type DEN-3 16562; DEN-2 / 4VP1 provides DEN-2 PDK-53V backbone and prM-E gene from wild type DEN-4 1036; and DEN-2 / WN-PP1 provides DEN-2 16681 backbone and prM-E gene from West Nile NY99. Other chimeras disclosed herein are indicated in the same manner.

In a specific example, the chimeras disclosed herein contain the attenuated DEN-2 virus PDK-53 genome as the viral backbone, wherein the structural protein genes encoding the C, prM, and E proteins of the PDK-53 genome or a combination thereof Substitution of the corresponding structural protein gene from the DEN-4 virus and another flavivirus optionally protected against such as a different flavivirus or a different dengue virus strain. A newly discovered flavivirus or flavivirus pathogenic bacteria can also be incorporated into the DEN-2 backbone.

In the non-structural protein region, the mutation of Gly to Asp (wild-type to PDK-53) was found at non-structural protein NS1-53 (genomic nucleotide position 2579); Leu became Phe (wild-type to PDK-53) The mutation was found at the non-structural protein NS2A-181 (genomic nucleotide position 4018); the mutation from Glu to Val (wild type became PDK-53) was found at the non-structural protein NS3-250 (genomic nucleotide position 5270) And a mutation from Gly to Ala (wild-type to PDK-53) was found at the nonstructural protein NS4A-75 (genomic nucleotide position 6599).

The attenuated PDK-53 virus strain has a mixed genotype at the nt 5270 genome. The majority (approximately 29%) of the virus population encodes the non-mutated NS3-250-Glu present in the wild-type DEN-2 16681 virus, rather than the NS3-250-Val mutation. Because both gene variants are non-pathogenic, this mutation is not necessary in non-pathogenic chimeras.

It has been found previously that the non-pathogenicity of the attenuated PDK-53 virus strain may be due to mutations in the nucleotide sequence encoding the non-structural protein and in the 5 'non-coding region. For example, single mutations at NS1-53, double mutations at NS1-53 and 5'NC-57, double mutations at NS1-53 and NS3-250, and NS1-53, 5'NC-57 and The three mutations in NS3-250 will reduce the toxicity of DEN-2 virus. Therefore, any genome containing this non-conservative amino acid substituted or nucleotide substituted dengue-2 virus at these loci can be used as a basis for obtaining the modified PDK-53 virus disclosed herein . If desired, another mutation in the stem / loop structure in the 5 'non-coding region can provide additional non-pathogenic phenotypic stability. Mutations in this region can disrupt possible secondary structures important for viral replication. In both DEN and Venezuelan equine encephalitis virus, a single mutation in this short (only 6 nucleotide residues in length) stem structure would disrupt the formation of hairpin structures. Additional mutations in this stem structure will reduce the likelihood of recovery at this locus while maintaining virus viability.

The mutations disclosed herein can be achieved by any method known in the art, including, but not limited to, site-directed mutagenesis, direct synthesis, deletion, or other methods known to those skilled in the art. Those skilled in the art will understand that the pathogenicity screening assays described herein and well known in the art can be used to distinguish between pathogenic and non-pathogenic skeleton structures.

    Construction of flavivirus chimeras    

The flavivirus chimeras described herein can be produced by splicing one or more structural protein genes of flaviviruses to which immunity is to be generated, into the PDK-53 dengue virus genome backbone, or by using other known techniques in this technology. The method uses recombinant engineering to remove the corresponding PDK-53 gene and then replace it with the dengue-4 virus gene or other genes known in the art.

Alternatively, the nucleic acid sequence of any of the constructs disclosed herein and the nucleic acid molecule encoding a flavivirus protein can be synthesized using any known nucleic acid synthesis technique and then inserted into a suitable vector. Therefore, the non-pathogenic and immunogenic viruses in the specific examples herein can be generated using recombinant engineering techniques known to those skilled in the art.

A target gene encoding a flavivirus structural protein of interest, DEN-4 (alone or in combination with another flavivirus), can be inserted into the backbone. The flavivirus (eg, dengue virus) gene to be inserted may be a gene encoding protein C, PrM and / or protein E. For example, a sequence inserted into the dengue-2 backbone can encode both PrM and E structural proteins, or only a single structural protein. The sequence inserted into the dengue-2 backbone can encode all and one of the C, prM, and E structural proteins.

Suitable chimeric viruses or nucleic acid chimeras containing nucleotide sequences encoding structural proteins of other flaviviruses or dengue virus serotypes can be screened for attenuated phenotypic markers that show non-pathogenicity and screened Wait for immunogenicity to assess whether it is available as a vaccine. The antigenicity and immunogenicity can be assessed by using conventional screening procedures known to those skilled in the art to respond to flavivirus antibodies or immunoreactive serum in test tubes and / or in vivo.

    Dengue virus vaccine    

In certain embodiments, chimeric viruses and nucleic acid chimeras can provide live attenuated viruses that can be used as immunogens or vaccines. Some specific examples include chimeras that exhibit high immunogenicity against dengue-4 virus without causing dangerous pathogenicity or death.

In order to reduce the incidence of DHF / DSS in individuals vaccinated against a vaccine against only one dengue virus serotype, it is necessary to provide a two-, three- or four-valent vaccine that can simultaneously immunize two to all four virus serotypes. The production of a tetravalent vaccine can be achieved by combining live attenuated dengue-2 (eg, dengue-2 PDK-53) with dengue-2 / 1, dengue-2 / 3, and dengue-2 / 4 as described herein. Chimeras in pharmaceutical carriers suitable for multivalent vaccine administration to combat all four dengue virus serotypes. Other formulations may include the above bivalent or trivalent formulations, where the formulation includes one or more novel DEN-4 chimeric constructs.

The chimeric virus or nucleic acid chimeras disclosed in some specific examples herein may include structural genes of wild-type or attenuated viruses in the pathogenic or attenuated DEN-2 virus backbone. For example, the chimeras can express structural proteins of the wild-type DEN-4 1036 virus and candidate vaccine derivatives in their respective DEN-2 PDK-53 backgrounds. In some specific examples, the pharmaceutical or experimental composition disclosed herein may include one or more constructs with the names DENVax-4e, DENVax-4g and / or DENVax-4h, alone or in combination with other flaviviruses. Constructs. In some examples, these constructs can be used with one or more viral seed bank (MVS) (e.g., DEN-1 / DEN-2) constructs disclosed herein. Other specific examples may include the DEN-4 constructs disclosed herein and other flavivirus chimeras, such as those made on the yellow fever framework or the West Nile framework or other flavivirus frameworks, where these flaviviruses are embedded The zygote can form a chimeric construct with a dengue virus serotype that, when introduced into an individual, can induce an immune response to the virus in that individual.

Viruses used in the chimeras described herein can be grown using techniques known in the art. Viral plaque titration was then performed and plaques were counted to assess the viability and phenotypic characteristics of the growth culture. Subculture of wild-type virus by culturing cell lines to derive attenuating candidate starting materials.

Chimeric infection clones can be constructed from a variety of available dengue serotype clones. If necessary, the selection of virus-specific cDNA fragments can also be performed. CDNA fragments containing structural or non-structural protein genes can be amplified from dengue virus RNA using various primers by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified fragments can be selected to break into other intermediate selection lines. Afterwards, intermediate and chimeric dengue virus clones can be sequenced to confirm the accuracy of the inserted dengue virus-specific cDNA.

In some specific examples, a complete genomic chimeric plastid constructed by inserting structural or non-structural proteins of dengue serotype virus into the vector can be obtained using recombinant techniques known to those skilled in the art.

    Analysis of nucleotides and amino acids    

In some specific examples, PDK-53 does not contain amino acid mutations in E protein relative to wild-type dengue-2 virus; DEN-1, DEN-3, and DEN-4 attenuated viruses may have Amino acid mutation. Wild-type DEN-3 16562 has been shown to contain a small number of variants that contain a T at nucleotide 1521, which will guide the incorporation of leucine at polyprotein position 476 of the E protein and amino acid residue position 476. Each of the latter three viruses may have a mutation in the E protein from Glu to Lys (the parent becomes a vaccine), but the mutation is at a different amino acid residue in the E protein. This substitution results in a conversion from a negatively charged amino acid to a positively charged amino acid. The substitution of Glu in the E protein of the DEN-4 vaccine virus to Lys is the only mutation in the E protein, and the E protein of the DEN-1 and DEN-3 vaccine viruses has five and three amino acid mutations, respectively.

In some specific cases, the NS1-53 mutation occurred in the DEN-2 PDK-53 virus, and the attenuated phenotype of this virus is important because the NS1-53-Gly of the DEN-2 16681 virus The flaviviruses (including tick-borne viruses) are conserved. The DEN-4 viral construct disclosed herein may contain an amino acid mutation at position 253 of the NS1 protein. This locus, which is a mutation of Gln to His in the DEN-4 PDK-48 virus, is Gln in all four dengue virus wild serotypes. This Gln residue is unique among dengue viruses in the flavivirus genus. The NS1 protein is a glycoprotein that is secreted by flavivirus-infected cells. It is present on the surface of infected cells, while NS1-specific antibodies are present in the serum of individuals infected with the virus. There have been reports of protection of animals with NS1 protein or negative immunization with NS1 specific antibodies.

Certain mutations are found in the NS2A, NS2B, NS4A, and NS4B proteins of the attenuated DEN-1, -2, -3, and -4 strains, which are conserved in nature. Mutations in the NS4A-75 and NS4A-95 of the DEN-2 and DEN-4 vaccine viruses generally occur at conserved amino acid positions between dengue viruses, not between flaviviruses.

Flavivirus NS3 protein has at least two identifiable functions: viral protease and RNA helicase / NTP enzyme. The 698-aa long (DEN-2 virus) NS3 protein contains an amine terminal serine protease domain (NS3-51-His, -75-Asp, -135-Ser catalytic ternary group), followed by RNA helicase Sequence motifs for the / NTP enzyme function (NS3-196-GAGKT), -284-DEAH, -459-GRIGR (sequence identification number: 26), shown previously). In the NS3 protein of the DEN-1, DEN-2 or DEN-3 virus, no mutations occurred in the identifiable motifs. The mutation of NS3-510 Tyr to Phe in DEN-1 PDK-13 virus is a conservative mutation. Because wild-type DEN-2, -3, and -4 viruses contain Phe at this position, mutations from Tyr to Phe do not appear to be involved in attenuating the DEN-1 virus. In most mosquito-borne flaviviruses, the mutation of NS3-182 Glu to Lys in the DEN-1 PDK-13 virus occurs at a conserved position, such as Asp or Glu, and it may be involved in some attenuation. This mutation is located at 15 amino acid residues upstream of the helicase motif of GAGKT (Sequence ID: 27). In some dengue-2 viruses, NS3-250-Glu in DEN-2 16681 virus, with the exception of yellow fever virus, is conserved in all mosquito-borne flaviviruses.

The nucleic acid probes used in some specific examples herein will selectively hybridize to nucleic acid molecules encoding DEN-1, DEN-3, and DEN-4 viruses or their complementary sequences. "Selective" or "selectively" means a sequence that does not hybridize to other nucleic acids that would interfere with proper detection of dengue virus. Therefore, in the design of hybridized nucleic acids, selectivity depends on other components in the sample. The segment of the hybridizing nucleic acid and the nucleic acid to be hybridized should be at least 70% complementary. The term "selective hybridization" used to describe nucleic acids herein excludes occasionally random hybridized nucleic acids, and thus has the same meaning as "specific hybridization". The selective hybridizing nucleic acid disclosed herein can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% complementarity with the sequence fragment to be hybridized, preferably 85% Or higher.

Consideration of sequences, probes and primers that will selectively hybridize to a nucleic acid encoding the nucleic acid or its complementary or opposite strand. As long as the functional species-specific hybridization ability is maintained, and there are slight modifications or substitutions in the nucleic acid, specific hybridization of the nucleic acid can also occur. "Probe" means a nucleic acid sequence that can be used as a probe or primer that selectively hybridizes to a complementary nucleic acid sequence for their detection or amplification. The length of the probe can vary from about 5 to 100 nuclei. The nucleotides are preferably from about 10 to 50 nucleotides, or most preferably from about 18 to 24 nucleotides.

If used as a primer, the composition preferably includes at least two nucleic acid molecules that will hybridize to different regions of the target molecule in order to amplify the desired region. Depending on the length of the probe and primer, the range of the target region can be between 70% complementary bases and all complementary bases, and can still hybridize under severe conditions. For example, for the purpose of detecting the presence of dengue virus, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to be hybridized is at least sufficient to distinguish between hybridization with nucleic acid and other organisms.

A nucleic acid sequence encoding a DEN-4, DEN-3 or DEN-1 virus can be inserted into a vector such as a plastid, and then recombinantly expressed in a living organism to produce a recombinant dengue virus peptide and / or polypeptide.

    Nucleic acid detection method    

The present invention provides a rapid genetic test for diagnosing each of the vaccine viruses described herein. This specific example of the present invention improves the analysis of viruses isolated from vaccinated human serum that has progressed to viremia, and improves the characterization of viremia in non-human primates immunized with candidate vaccine viruses.

These sequences include a diagnostic TaqMan probe for reporting the detection of cDNA amplicons amplified from viral genomic RNA templates using reverse transcriptase / polymerase chain reaction (RT / PCR); and a design designed for amplification The forward and reverse primers of the cDNA amplicon are described below. In some cases, one of the amplified primers is designed to contain a vaccine virus-specific mutation at the 3 'terminal of the amplified primer, which can effectively make the testing of vaccine strains more specific because the primers at the target position Extension, so the amplification reaction only occurs when the viral RNA template contains the specific mutation.

An automated nucleic acid sequence detection system based on PCR can be used, which is becoming increasingly widely used in diagnostic laboratories. TaqMan analysis is a highly specific and sensitive analysis method that allows automatic real-time visualization and quantification of PCR-generated amplicons from a sample nucleic acid template. TaqMan determines the presence or absence of specific sequences. In this analysis, forward and reverse primers were designed to bind the upstream and downstream of the target mutation site, respectively. The specificity detection probe is designed to have a melting point temperature of about 10 ° C higher than each amplification primer, and contains a specific mutation of a vaccine virus nucleotide or its complement (depending on the RT / PCR amplicon strand to be detected). (Determined), constituting the third primer component of this analysis. Probes designed to specifically detect a mutant locus in one of the chimeric constructs may contain specific nucleotide changes for the detection of any mutation.

One strategy for diagnostic genetic testing uses molecular beacons. The molecular beacon strategy also uses primers for RT / PCR to amplify amplicons, and probes that contain reporters and quencher dyes at the probe ends to detect specific sequences within the amplicon. In this analysis, the probe forms a stem-loop structure. The 5'- and 3'-terminal reporter dyes and quenching dyes are respectively located at the terminals of the short stem structure, which bring the quenching dye to the vicinity of the reporter dye. The stem-loop structure melts during the denaturation step of the RT / PCR analysis. If the target viral RNA contains the target sequence and is amplified by forward and reverse amplification primers, the open loop of the probe will hybridize to the target sequence during the cycling adhesion step. When the probe is attached to each strand of the amplicon template, the quencher and the reporter dye are separated, and the fluorescence of the reporter dye can be detected. This is an instant identification and quantitative analysis method, which is very similar to the TaqMan analysis method. Molecular beacon analysis uses different quenching and reporter dyes than those used in TaqMan analysis.

    Pharmaceutical formula    

Any pharmaceutical formulation known in the art of vaccines will be considered here. In certain embodiments, the formulation may contain DEN-4 constructs alone, or DEN-4 constructs in various ratios and one or more additional DEN serotypes (or other flavivirus compositions), as disclosed herein DEN-4 structure, which depends on the dengue virus subtypes expected to be exposed or present in the endemic area. The formulation may be considered to contain other agents for vaccinating individuals, including, but not limited to, other active or inactive ingredients or compositions known to those skilled in the art. In certain embodiments, adjuvants may be included in the formulations disclosed herein.

Other aspects of the invention may include modulating the immune response of a vaccine against dengue virus in an individual. A vaccine against dengue virus may include a composition comprising a ratio of dengue virus, live attenuated dengue virus, or a fragment thereof, such as a protein or nucleic acid derived or obtained from a dengue virus serotype. The ratio of the various serotypes may be equal, or some serotypes may be present at a higher rate than others, depending on the demand or exposure or possible exposure to the virus. According to these specific examples, the ratio of any of serotypes 1, 2, and 3 to the DEN-4 construct disclosed herein can be 1: 2, 1: 3, 1: 4, 1:10, 1 : 20; 1: 1: 1: 1, 1: 2: 2, 1: 2: 1, 1: 1: 1: 1: 1, 1: 2: 1: 2; 1: 3: 1: 3, 2: 3: 3 : 3, 5: 4: 5: 5, 4: 4: 4: 5, 1: 2: 2, 4: 4: 5: 5, 4: 4: 5: 6, or any ratio, depending on, for example, The number of serotypes present in the formulation, the intended response, and the desired effect. The last number represents the amount of DEN-4 in the formulation. Each number represents a magnification of 10 (6 = 10 ⁶ PFU). Any dengue virus serotype formula can be considered for the production of vaccines (e.g., attenuated viruses, etc.) for administration to individuals in need.

The specific examples herein provide a biocompatible form suitable for in vivo administration, and administration of the composition to an individual. "Biocompatible form suitable for in vivo administration" means the form of the active agent to be administered (eg, a specific example of a pharmaceutical protein, peptide or gene, etc.), wherein the therapeutic effect of the active agent is superior to Any toxic effects. A therapeutic composition administered in a therapeutically active amount is defined as the effective amount at the dose and time required to achieve the desired result. For example, the amount of therapeutic activity of a compound can vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in the individual. The dosing regimen can be adjusted to provide the optimal therapeutic response.

In a specific example, the compound (e.g., the pharmaceutical protein, peptide, etc. of the specific example) can be administered by conventional methods, such as subcutaneous, intravenous, intradermal, oral administration, inhalation, transdermal administration, skin Internal, vaginal, topical, intranasal or rectal administration. Depending on the route of administration, the active compound may be coated with a material to prevent the compound from being degraded by enzymes, acids, and other natural conditions that cause the compound to be inactive. In a specific example, the compound can be administered intranasally, such as by inhalation.

The compound can be administered to an individual in a suitable vehicle or diluent, together with an enzyme inhibitor, or to an individual in a suitable vehicle such as microlipid particles. The term "pharmaceutically acceptable carrier" as used herein is meant to include diluents such as saline and aqueous buffer solutions. It may be necessary to coat the compound with the material or co-administer the compound with the material to prevent it from losing activity. The active agent can also be administered parenterally or intraperitoneally. It can also be prepared in glycerin, liquid polyethylene glycols and mixtures thereof, and dispersants in oils. Under ordinary conditions of storage and use, these products may contain a preservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injection use can be administered by methods known in the art. For example, sterile aqueous solutions (herein water-soluble), or dispersants and sterile powders for the temporary preparation of sterile injectable solutions or dispersions can be used. In all cases, the composition is sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. A pharmaceutically acceptable carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, using a coating such as lecithin; maintaining a desired particle size in the case of a dispersant; and using a surfactant to maintain proper fluidity. Microbial prevention can be achieved by heating, exposing the agent to cleaning agents, radiation, or adding various antibacterial or antifungal agents.

When required, when preparing sterile injectable solutions, the required amount of active agent can be combined with one or a combination of the ingredients described above.

The aqueous composition may include an effective amount of a therapeutic compound, a peptide, an epitope core region, a stimulant, an inhibitor, and the like, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The compounds and biological materials disclosed herein can be purified by methods known in the art.

When formulated, the solution should be compatible with the formulation of the dosage form and administered in a therapeutically effective amount. The formulation can be easily administered in different dosage forms, such as the type of injection solution described above. The use of low-release capsules, regular release of microparticles, and the like can also be considered. These special aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

The active therapeutic agent can be formulated in a mixture so as to contain from about 10 ² to about 5 × 10 ⁶ PFU of each of the constructs considered herein. It is also possible to administer a single dose or multiple doses in a suitable time schedule under predetermined conditions. In some specific cases, in order to elicit an immune response with less interference or different lymph nodes, a double dose is administered at a single anatomy or multiple anatomic locations on day 0. In certain embodiments, a single-, two-, three-, or four-valent formulation of a dengue virus construct can be administered to an individual. Any of these formulations can be provided to an individual in a single or multiple doses. In some specific cases, one dose can be administered and added after a period of time.

In another embodiment, nasal solutions or sprays, aerosols or inhalants can be used to deliver compounds of interest. Additional formulations suitable for other modes of administration include suppositories and pessaries. Rectal pessaries or suppositories can also be used. Generally in the case of suppositories, traditional binding agents and carriers may include, for example, polyglycols or triglycerides; such suppositories may contain actives ranging from 0.5% to 10%, preferably 1%, 2% A mixture of ingredients is formed.

Pharmaceutical compositions containing α1-antitrypsin, its analogues or inhibitors of serine protease activity or functional derivatives thereof, for example, subcutaneous, intramuscular, intranasal, oral, topical, transdermal, parenteral , Gastrointestinal tract, transbronchial, and alveolar methods, administered to individuals, especially humans.

In certain embodiments of the method of the invention, the individual can be a mammal such as a human or vertebrate and / or a mammal.

A specific example method of the present invention provides vaccination for individuals who are preparing to travel to countries with dengue virus. In other specific examples, the individual may be a resident within an endemic area. One or two shots can be considered on day 0 and then added after less than 30 days, 2 months, 3 months, 6 months, or almost 1 year.

    Set    

Other specific examples relate to the use of the methods disclosed herein (e.g., methods of administering or administering vaccines) and sets of compositions. Some specific examples pertain to sets of vaccine compositions that are used to prevent or treat individuals who have, are exposed to, or are likely to be exposed to one or more dengue viruses. In certain embodiments, the kit may contain one or more formulations of dengue virus serotypes (eg, attenuated vaccines). Kits can be portable, for example, capable of being transported to and used in remote areas such as military bases or remote villages. Other sets can be used in health facilities to treat individuals who have been exposed to one or more dengue viruses or who may be at risk for dengue virus.

Kits may also include suitable containers, such as vials, vials, mini- or microcentrifuge tubes, test tubes, flasks, vials, syringes, or other containers. When an additional component or agent is provided, the kit may contain one or more additional containers for containing the agent or component. The kits herein may also typically include components for tightly containing the agent, composition, and any other reagent containers for commercial sale. Such containers can include injection or blow molded plastic containers in which desired vials can be stored. Optionally, one or more additional agents such as an immunogenic or other antiviral, antifungal, or antibacterial agent may be required for the composition, for example, to combat one or more additional microorganisms Composition of the vaccine.

The following non-limiting examples will further illustrate specific examples of the present invention, and in any case, they should not be construed as being limited in scope. Those familiar with this technique should understand that the technology disclosed in the example is considered to be a better model of this practice because it is found to be a good technology that can work well in the example disclosed here. However, according to this disclosure, those skilled in the art should understand that there can be many changes in the specific specific examples disclosed, and still obtain the same without departing from the technical ideas and scope here. Or similar results.

    Paradigm         Example 1    

In certain exemplary methods, the DEN-4 chimera construct is formulated as a pharmaceutically acceptable composition.

Figure 1A depicts the skeleton of an exemplary virus disclosed herein as a specific example, called the DEN-2 PDK-53 genome (disclosed previously in PCT / US01 / 05142, U.S. Patent Application No. 10 / 204,252. The entire contents of various purposes are incorporated herein by reference, which can be used to generate modified DENV-4 constructs (also referred to as the second generation). As shown in Figure 1A, specific amino acid substitution mutations in non-structural regions such as NS1-G53D, NS3-E250V, and C57T in the 5 'non-coding region in the DEN-2 PDK-53 genome have been identified In the nucleotide substitution mutation. The original DENV-1, 3, and 4 constructs (also referred to as the first generation) were generated by using the structural coding sequences of the respective serotypes to replace the coding sequences of prM and E in the DENV-2 backbone (Figure 1B). In order to enhance the replication efficiency of modified DENVax-4 constructs in vitro and in vivo, and to improve the immunogenicity in the host, the non-coding region, structural protein coding region and / Or non-structural regions introduce point mutations. For example, modifications can be made upstream of the amino acid sequence of the current DENVax-4 C / prM break site to simulate wild-type dengue-4 (which DENVax-4 currently contains) as opposed to dengue-2.

Some additional modifications are provided in Table 1 in certain exemplary DENV-4 constructs. Described below are sequences aligned in certain modified DENVax-4 constructs (DENVax-4b, DENVax-4c, and DENVax-4d) to DENV-2, DENV-1, and the original DENVax-4 wild-type sequence. Selected changes in these sequences are marked in bold and subscripted:

Figure 4 shows a schematic diagram of an original and modified DENV-4 construct. DENVax-4e is generated by using the DENVax-4b sequence as a backbone, and then incorporating C into Y at the amino acid 107 of the capsid protein. DENVax4f, DENVax4g, and DENVax4j are based on the DENVax-4a backbone, but contain a wild-type response to the attenuated mutation of the PDK-53 sequence indicated in Figure 4: DENVax-4f contains wild-type (DEN-2 16681) NS2A and NS4A; DENVax-4g contains wild-type 5'NCR, NS2A, and NS4A; and DENVax-4j contains wild-type 5'NCR. DENVax4h has a substitution of E to K at mantle 417 in the DENV4 1036 sequence. DENVax4i has a substitution of M to L at NS4A. DENVax4k has a PrM / E gene from DENV-4 H241 which replaced the 1036 strain (Table 1). Mutations constructed in DENVax4e, h, and i are based on sequence data derived from successive generations of DENVax4 and DENVax-4b in Vero cells.

DENVax-4b contains all 7 amino acid changes, DENVax-4c contains all 9 amino acid changes, and DENVax-4d contains a monoamino acid deletion, which causes the framework of this sequence to move. RNA is transcribed and electroporated into Vero cells for expansion and virus reactivation. To test the growth efficiency of DENVax-4b and DENVax-4c in Vero and C6 / 36 cells in terms of growth duration experiments, DENVax4-P2, DENVax4-P8 and DENVax2-P2 were used as control groups. DENVax-4-P2 and DENVax-2-P2 are virus samples that have not been genetically selected for virus plaque purification. The method is to select individual virus plaques from a single layer of DENVax-infected Vero cells, and then place a layer containing Agar gel of sexual red in order to visualize a single plaque. DENVax-4-P8 has been plaque-purified to obtain a virus storage strain of the selected DENVax-4 genotype. This procedure was performed to generate seed protoviruses (MVS) without reverted attenuating mutations. After growing in Vero cells, DENVax-4c does not reach a sufficient peak titer and has a slower initial growth rate than DENVax-4 or DENVax-4b. There was no significant difference between the peak titers of the growth curves of DENVax-4 and DENVax-4b in Vero cells, and both had similar initial growth rates. In the analysis of C6 / 36 mosquito cells, both the growth of DENVax-4b and DENVax-4c reached a peak titer significantly smaller than that of wild-type DENV-4, confirming their reduced toxicity.

    Example 2         Generation of entire infectious cDNA clones    

To generate entire infectious cDNA clones containing modified DENVax-4 construct sequences, a multi-step digestion / ligation scheme was used. It has the following steps: 1) Insert an AgeI / MluI synthetic fragment including a modified nucleic acid sequence or a modified structural protein sequence (e.g., b, c or d, or any of the above constructs) into pD2 / 3-PP1-5 ', producing pD2 / 4i-b, c or d; 2) digesting the entire pDENVax-4 cDNA clone, extracting the MluI / NgoMIV fragment, and inserting it into pD2 / 4i-b, c or Obtain pD2 / 4i-b, c or d at the corresponding position in d; 3) Digest pDENVax-4 cDNA clones, extract NgoMIV / XbaI fragment, and insert it into pD2 / 4i-b, c or d In the corresponding position, the entire infectious selection strain containing the modified sequence is generated. Using sequence analysis, the sequence of the last infectious colony was confirmed.

    Virus generation    

A cDNA clone of each modified construct was transcribed into genomic virus RNA. Using electroporation, RNA was transformed into Vero cells. The virus was allowed to grow for 12 days while CPE was monitored and then collected. The first collection after electroporation is called P1 (subculture 1). Subsequent amplifications and subsequent generations are referred to as P2, P3, and so on. The original DENVax-4 and DENVax-4b and c had limited CPE, however DENVax-4d did not produce any obvious CPE. Unlike other viruses growing in test tubes, dengue virus does not produce very much CPE when grown in Vero cells. Although the DENVax-4d virus does not produce any CPE in a test tube, it simultaneously expands with other strains. The amplified P1 virus (eg, DENVax-4b, c, d P1) produced high titers for sequence analysis and growth curve experiments. DENVax-4d did not show titers, so no virus was produced after electroporation.

The DENVax-4b and -4c viruses have been completely sequenced. The DENVax-4b-P2 virus has two mutations. Nucleotide 416 is at the genetically modified position in the capsid (close to the C / prM linker) of the DENVax-4b virus. Because this is a mixed population, nt 416 reverts to an "A" nucleotide instead of a genetically engineered "G" nucleotide, resulting in the expected amino acid arginine being replaced with serine. The second mutation line found in DENVax-4b-P2 is at nucleotide 8769. This results in an amino acid change from the expected glutamic acid to proline. This mutation is in the NS5 gene region of the infectious selection strain. The DENVax-4c-P2 virus has four mutations. They all responded completely, unlike DENVax-4b with mixed ethnicity. The mutation at 400 nucleotides in the capsid region of the genome results in genetic engineering modification at the C / prM linker. This results in that the amino acid expected at this position is proline, not threonine. The other three mutations are in non-structural genes, two of which cause amino acid changes, and one is a silent mutation.

    Viral phenotype and gene characterization    

The phenotypic characterization of the virus was performed in Vero cells and C6 / 36 (Alternaria albicans) cells. Figures 5-6 show exemplary surface characteristics of virus variants produced by modified DENVax-4 constructs. Plaque size is an important attenuating marker and can be used to analyze every virus variant. As shown in FIG. 5, the plaque diameter of the DENVax-4b virus was approximately 0.3 cm. The plaque diameter of DENVax-4c virus was about 0.1 cm. The plaque diameter of DENVax-4h virus was about 0.3 cm. The plaque diameter of DENVax-4i virus was about 0.1 cm. The plaque diameter of DENVax-4c virus was about 0.2 cm. Each size represents the average of 5-10 plaques. Plaques in candidate viruses b and c, especially DENVax-4c, are not uniform. There are mixed populations of small and some large plaque viruses. The possible nucleotide changes of the sequence variability of these small and large plaques can be described, which may help attenuate and adapt in vitro. If desired, these mutations can be genetically engineered into additional DENVax-4 variants. Figures 6A-6F show exemplary results of each of wild-type DENV-4 virus (A) and DENVax-4b-f virus variants (B-F).

In some exemplary methods, the growth curve of the virus produced by the DENV-4 construct is determined. Monolayer Vero cells were infected with various DEN-4 construct compositions (e.g., DENVax-4b-j and DENVax-4P1) at a MOI of 0.001. In some demonstration experiments, by the 13th day, samples are taken every other day and a sample volume is titrated. The DENVax-2-P2 virus reached its peak titer relatively quickly. The peak titer and growth rate of DENVax-4b virus are similar to those of DENVax-4-P2 and P8 viruses. The DENVax-4c virus initially had a slower growth rate, but eventually reached a similar peak titer. The efficiency and peak titer of DENVax-4b and DENVax-4c viruses are comparable to the original DENVax-4. In other demonstration experiments, samples are taken every other day from day 2 to day 12. Culture media obtained on days 7, 9, and 11 were retained and stabilized for further study. Samples from day 2 to 12 were titrated with IFA. The DENVax-4e-4h virus showed similar peak titers as the control DENVax- (Figure 7).

To demonstrate the attenuating effect, the replication efficiency of each vaccine virus in C6 / 36 mosquito cells should be reduced compared to the wild-type virus. This phenotype is a substantially safe feature of the DENVax vaccine virus to ensure that there is no possibility of transmitting a naturally attenuated chimeric virus. In some exemplary methods, growth in C6 / 36 mosquito cells is performed to compare the growth characteristics of DENVax4b and DENVax-4c viruses to wild-type dengue 4 virus (strain 1036). In other demonstration experiments, comparisons between DENVax4e-4j and the original DENVax4 were performed. C6 / 36 cells in duplicate bottles were infected with each P2 virus (-b, -c, and wild type) at a MOI of 0.001, and then grown for 14 days. Dengue 4 wild-type virus (WT D4 1036) replicates most efficiently and has the highest titer. DENVax-4b virus replicated very well in C6 / 36 cells, reaching a peak titer of 2.7 x 10 ⁶ pfu / mL on day 14. The DENVax4c virus grew very slowly. After day 6, it accelerated to day 14 and reached a peak titer of 2.2 x 10 ⁴ pfu / mL. On day 6, both DENVax-4b and DENVax4c had similarly reduced growth compared to the wild type in C6 / 36 cells, and had similar potencies as the original DENVax-4. In other demonstrative experiments, the chimeric dengue virus strains of some specific examples here are grown in C6 / 36 mosquito cells. DENVax4e-4j growing in mosquito cells was compared with control DENVax4. C6 / 36 cells in duplicate were infected with each P2 virus with an MOI of 0.001, and then grown for 12 days. Samples were collected from day 2 to day 12. Compare growth in mosquito cells to Vero cells. The culture medium was taken and stabilized on the 7th, 9th, and 11th days. Samples from day 2 to day 12 were titrated with IFA and analyzed for virus yield. The virus titer and the growth of the construct were compared with the control DENVax4 (see Figure 8).

Figures 7-8 show exemplary diagrams illustrating the growth curves of various DENV4 construct viruses in Vero cells (Figure 7) and C6 / 36 mosquito cells (Figure 8). Many of the novel DENVax4 constructs in some specific examples here will produce higher titers in Vero cells than the control DENVax4 (Figure 7), while in mosquito cells will produce lower titers than the control DENVax4 (Figure 8) , As desired for further evaluation in animal models (see Table 2).

    Safety and efficiency of virus variants in mice    

Studies were performed to analyze the immunogenicity of DENVax-4b, c or other variants. Table 2 presents an exemplary study design. A group of 10 AG129 mice were subcutaneously (in the palm rest) inoculated with 10 ⁴ PFU of each original DENVax-4 and different mutant strains. The control group was injected with a solution containing only excipients. These mice received additional doses after 42 days. Serum samples were taken for serum analysis on days 42 and 56 after the first vaccination, and PRNT was used to analyze seroconversion. When the second-generation DENVax-4b and c variants were used as immunogens, the immunogenicity of DENVax-4 was not significantly improved.

To further modify and expand the immunogenicity of DENVax-4b, the virus was adapted to grow on Vero cells in a test tube. This is carried out for 10 generations of blind transmission in Vero cells. The theory is that adaptation in mammalian cell culture will strengthen the virus's replication ability and therefore enhance its immunogenicity. This "new" second-generation DENVax-4 is called DENVax-4b-P10 (succession-10).

Another evaluation was performed including studies of different four-valent formulations of dengue virus vaccines for three different DEN4 viruses; first generation, second generation (4b construct), and homologous wild-type DEN4 1036. A group of 6 mice injected subcutaneously, containing 10 ⁴ PFU DENVax-1, 10 ³ PFU DENVax-2, 10 ⁴ PFU DENVax-3, and 10 ⁵ PFU DENVax 4 (4: 3: 4: 5) with a volume of 50 μL Tetravalent vaccine formulation. Control mice were injected with 10 ⁵ PFU of the first, second, or wild-type 1036 monovalent DENV4 in the same way. Groups of mice injected with diluent only served as a control group. All mice received additional injections of the corresponding vaccine formulations on day 42 and blood was collected on days 0, 21, 41, and 56 after the first vaccination to assess resistance to all four DENV serotypes And whether antibodies appear. On day 56, mice of groups 4, 5, and 6 were challenged with DENV-2 (NGC strain) to assess survival (no mice are now adapted to the lethal DENV4 strain for effective analysis). As shown in Table 3, the first-generation and second-generation DENVax-4 vaccines, when administered alone, have comparable immunogenicity curve. However, when they are provided as a quadrivalent dengue virus vaccine, the immune response to DENV-4 will be reduced only by interference with the second-generation DENVax-4. The wild-type DENV4 line is highly immunogenic, and in the case of a tetravalent dengue virus vaccine, it interferes with and inhibits the response of neutralizing antibodies induced by the other three dengue virus vaccines. Wild-type DENV4, 1st and 2nd generation DENVax-4, provide protection against the challenge portion of heterologous DENV-2.

    Security and efficiency of 2nd generation DENVax in NHP    

Immunogenicity of DENVax in a group of four Cynomologus macaques was evaluated after subcutaneous injection of each tetravalent DENVax formulation with a 0.5 Ml injection volume (Table 4 and Figure 2). The purpose of this study was to evaluate important second-generation DENVax-4 constructs compared to first-generation DENVax-4. Additionally, different approaches were explored to improve the neutralizing antibody response to DENV-4 induced by DENVax. These include testing formulations containing 10X doses of DENVax-4 (known as "new formulations") as previously used, as well as reducing the DENVax-2 component of the tetravalent mixture, as this vaccine is the most effective of the four vaccine virus strains Immunogenic. Test different days of administration, compare inoculation and supplementation on the same day (day 0), and inoculation on day 0 and supplementation on day 60. Serology samples were taken on days 0, 28, 58, 58 and 90. The kinetics of neutralizing antibody titers against the DENV-4 mutant virus and the original virus are shown in FIG. 3, and as an example, similar levels can be observed here.

The vaccine formulations for the NHP study were prepared in batches. Cohort 1 received a vaccine manufactured by cGMP that is consistent with vaccines currently being tested in human clinical trials. The NHP vaccine is provided and then back-titrated to determine the actual dose. These results are presented in Table 5 below.

After the first dose, samples of -11 (baseline), 3, 5, 7, 9, 11, 13, 15, 17, 21, 28, 58, 62, and 66 were taken for viremia studies. RNA was extracted from the serum samples, and then the virus titer was determined by a tetraplex qRT-PCR analysis. The only virus that provided any measurable viremia was DENVax-2, and this resolved on day 21 after the first vaccination. No virus was detected after the additional dose of vaccine was administered.

Serological evaluation was performed using a high-throughput PRNT assay in a 96-well culture dish. The comparison of the first and second generations of DENVax-4 (comparison of cohort 1 and cohort 2) did not show significant differences in the immunogenicity of these two viruses (Table 6).

Assessing higher doses of DENVax-4 (Group 3) shows that significant differences can be obtained in the kinetics of the DENV-4 neutralizing antibody response. When comparing two different vaccine formulations, the peak titers remained approximately the same (within a 2-fold dilution range), but the rate of peak titers obtained for DENV-4 was much earlier than the higher dose of DENVax-4 for immunization The rate. In addition, when the amount of DENVax-2 was reduced in the formulation (group 5), there was no significant difference in the kinetics of DENV-4 and the peak titer in serum response in this tetravalent DENVax vaccine.

FIG. 9 shows a flowchart of an exemplary procedure for testing the safety and efficiency of the viral constructs disclosed herein in animal models. For example, a composition comprising one or more chimeric dengue viruses used as a vaccine, such as a composition of DENVax4e, DENVax4f or DENVax4h or other chimeric constructs, or a control composition can be tested in an animal model such as AG129. On day 1, the animals are vaccinated and then supplemented with the same or different dengue virus vaccine compositions on or before day 30 or 42 or other appropriate time. The animals were then challenged by exposure to one or more dengue virus serotypes to assess the immune response induced by the challenge. Blood samples are collected, for example, on days 0, 30, 41, 48, 52, or 84 or other appropriate days to test for neutralizing antibodies, and on days such as 5 and 47 The other days of the day were tested for viremia (or deemed appropriate for the composition selected and the dosing procedure).

    Virus breeding and return to P1    

Plastid mutation generation was used to make new DEN-4 chimeric selection strains for the vaccine compositions disclosed herein. The primers were synthesized for point mutations and then used to amplify whole new infectious clones. The plate was digested with DpnI and the subsequent plastids were sequenced. RNA was transcribed and electroporated into Vero cells to produce a virus (P0) with a degree of passage 0, which was then amplified using a single passage (P1) in Vero cells.

Modifications at the shell / PrM linker in DENVax-4b clones do not appear to cause an increase in the immunogenicity of DEN-4 in NHP studies. Using data from subsequent generations of the DEN-4 and DENVax-4b blind series, three point mutations were identified that increase Vero cell adaptation. In addition, some attenuating mutations revert to wild-type sequences, increasing immunogenicity in mice. As shown in Figure 6, the larger IFU size of DENVax-4e and DENVax-4h and the phenotype that promotes cell lysis suggest that any of these selected strains can show increased immunity in mice . Experiments were performed to analyze the growth kinetics of each of the new DENVax-4 clones to determine if the inserted modifications would increase the adaptive effect of Vero cells. Tests were also performed in A129 mice to analyze immunogenicity.

    Growth kinetics    

The serial succession of dengue vaccine strains in Vero cells is a typical method for selecting strains more suitable for growth in test tubes. In these exemplary growth experiments, DENVax-2 (Figure 1) served as the control group, with the highest starting titers on day 2, followed by DENVax-4b-P10, DENVax-4, and DENVax-4b-P1. At the end of the growth period (day 12), DENVax-2 had the highest peak titers, followed by DENVax-4b-P10, DENVax-4b-P1, and DENVax-4.

    Amino acid changes in the sequence    

DENVax4-P10 genomic sequencing revealed mutations corresponding to amino acids E-417 EK (Glu-Lys) and NS4A-17 ML. The DENVax4b-P10 sequence shows a mutation corresponding to the amino acid C-107 CY. The E-417 EK mutation alters the amino acid residues, so that the amine group (NH ₂ ) replaces the carbonyl hydroxyl group. However, the R group is still charged and remains hydrophilic. The NS4A-17 ML mutation results in the removal of sulfate from the R group, but retains the non-polarity of the hydrophobic amino acid. The C-107 CY mutation results in a drastic change in the R group. Cysteine has an SH group, which is capable of forming a disulfide bond, whereas tyrosine has a carbon benzene ring containing a hydroxyl group. This causes the amino acid residue to become hydrophilic instead of hydrophobic, affecting its interaction with other amino acid R groups.

Figure 20 is a graph showing the titer of a DENVax-4 construct during a growth kinetic experiment. The day when each sample was taken is plotted on the x-axis, and the titer is plotted on the y-axis.

    NHP neutralizing antibodies against DENVax    

In some methods, studies of larger immunogenicity protocols are used to evaluate DEN-4 chimeric constructs. DENVax-4b was tested for its immunogenicity compared to DENVax-4a. On day 0, two doses were inoculated on cohorts 1 and 2 without additional vaccination. Use equivalent DENVax-4 (diamond) or DENVax-4b (square) in all doses. No significant difference in the geometric mean titer (GMT) of neutralizing antibodies was found between any of the serotypes including DENV-4. This shows that the use of DENVax-4b in a tetravalent DENVa dose does not affect or increase the anti-DENV-4 neutralizing antibody response. Figure 21: Comparison of GMT values for Group 1 (DENVax-4, diamond) and Group 2 (DENVax-4b, square). The GMT value is a measurement of the neutralizing antibody value derived from the plaque reduction neutralization technique.

Compared with the neutralizing antibody responses in group 1 and group 3, it was determined whether increasing the dose of DENVax-4 in tetravalent DENVax would increase the anti-DENV-4 immunogenicity. The results showed that primates immunized with higher doses of DENVax-4 showed increased GMT of primary neutralizing antibodies detected in the first 60 days compared to those immunized with conventional tetravalent DENVax. There were no significant differences in GMT between the other DENV serotypes (Figure 22). This shows that higher doses of DENVax-4 improve the anti-DENV-4 neutralizing antibody response.

The neutralizing antibody responses in groups 1 (diamonds) and 4 (squares) were compared in order to test the effect of the immunogenicity procedure on immunogenicity (Figure 23). Figure 23 illustrates GMT values demonstrating the effect of vaccine procedures on neutralizing antibody responses. This indicates that 2 doses on day 0 without addition, had no significant effect compared to 1 dose on day 0 and then 1 dose on day 60. Finally, compare the data from group 4 (diamond) and group 5 (square) to see if reducing the dose of DENVax-2 will affect the immune results of DENVax-4. The results showed that there was no significant difference in anti-DENV-4 GMT, but in anti-DENV-2 GMT, group 5 was significantly reduced. This indicates that the antibody response induced by DENVax-2 has no serious effect on the antibody response induced by DENVax-4 (Fig. 24). Figure 24 shows GMT values for Group 4 (diamond) and Group 5 (square) for all DENV serotypes. Results were measured using plaque reduction neutralization techniques.

The results of these experiments support that the constructs disclosed herein improve the response to DENV-4 neutralizing antibodies. In the formulation, increasing doses of DENVax-4 did show a significant increase in neutralizing antibody products. DENVax-2 does not seem to have an impact on the DENVax-4 neutralizing antibody response. In the method of two doses of the first dose and the method of the first dose and the supplementary inoculation, there are only slight differences to no difference in the antibody production. Produces sufficient anti-DENV-4 1036, which is the strain currently used in DENVax-4, to neutralize antibody titers. A successful DENV-4 vaccine should be able to properly neutralize multiple wild-type DENV-4 strains, including newly evolved strains with genomic modifications, different genotypes, and different phenotypes.

During sequencing, three point mutations were identified between generation 1 and generation 10 in the two constructs. As discussed previously, these mutations are located in the capsid region of DENVax-4b, as well as in the prM and mantle genes of DENVax-4. Incorporating these mutations into the construct can provide increased growth in Vero cells by reducing the attenuating effect of the virus, which can improve immunogenicity. As disclosed herein, DENVax-4h has a 2-fold increase in neutralizing antibody titer, and DENVax-4e has a 1.5-fold increase in neutralizing antibody titer. In other methods, the increased immunogenicity of DEN-4 is tested and compared to other bivalent, trivalent, and tetravalent construct compositions.

    Materials and methods         Cell culture    

The Vero cell line is a mammalian cell derived from the US green monkey kidney. Vero cell lines are used in experiments in test tubes. Vero cells can be modified with Dulbecco's modified Eagle's medium (DMEM, Mediatech Inc., Manassas VA) at 37 ° C, supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan UT), 2% L-glutamine (Hyclone) and 1% Penicillin-Streptomycin (Pen-Strep, Hyclone). To subculture the cells, the cells were removed from the bottle surface using Tryple Express solution (Life Technologies, Grand Island NY).

    Viro Infects Monolayer    

About 48 hours before infection, Vero cells were seeded in T-75 cm ² bottles. DMEM supplemented with 10% FBS, 2% L-glutamic acid, and 1% penicillin-streptomycin was used as a cell growth medium. When cells were confluent, 4 mL of a 0.25% trypsin solution diluted 1: 5 in PBS was used to trypsinize 1 bottle. Count cells and establish MOI. The remaining two bottles, under the predetermined MO1, have DENVax-4-P2 (1st generation DENVax-4) or DENVax-4b-P3 or other constructs in 1mL, and are diluted in BA-1 dilution (bovine serum protein 1X) M199, 0.05M Tris-HCL, 1X L-glutamine, 7.5% sodium bicarbonate, 1X Pen-strep, 1X Fungizone). Use vibration every 10 minutes to avoid cell monolayer drying and allow virus to absorb onto Vero cells for 90 minutes. After absorption, 20 mL of DMEM supplemented with 5% FBS was added to each bottle. The bottles were incubated at 37 ° C for 7 days.

    Virus collection and subsequent infections: blind generation    

After a predetermined period, the CPE was observed on each bottle, and then the virus supernatant was collected and stabilized in 20% FBS for storage at -80 ° C. With 1 mL of the virus supernatant obtained from the aforementioned bottle, a Vero bottle confluent T-75 cm ² was inoculated before infection. Using vibration every 10 minutes, the virus was absorbed for 90 minutes. After the virus was absorbed, 20 mL of DMEM 5% FBS was added to each bottle. Every 7 days, new uninfected control bottles were streaked on petri dishes. Repeat this method every 7 days, lasting 10 weeks, each virus produces 10 generations, providing DENVax-4-P2-P1 to P10 or DENVax-4b-P3-P1 to P10, or other instructions for various constructs Title.

    Virus plaque titration    

Plaque titration of samples taken from DENVax-4-P2 and DENVax-4b-P3 and other chimeric constructs at weeks 1, 5, and 10 was performed to measure titers. The virus samples were sequentially diluted to 1x10 ^-1 to 1x10 ^-6 in BA-1 dilution. The plaque titration of the sample was performed in triplicate, and 100 uL of each dilution was adsorbed to a 6-well culture dish pre-seeded with Vero cells using shaking every 8 minutes for 90 minutes. After adsorption, the wells were covered with 4 mL of BSS / agar (NaCl, KCl, NaH ₂ PO ₄ -H ₂ O, glucose, CaCl ₂ -2H ₂ O, MgSO ₄ -7H ₂ O) solution, and then incubated at 37 ° C for ₄ hours. day. On day 4, the wells were covered with 2 mL of BSS / Agar / Neutral Red solution and then incubated overnight at 37 ° C. Plaques were counted on days 5, 6, and 7.

    Growth curve analysis    

Growth curves were established on Vero cells to analyze the growth kinetics of the adapted strains. As before, inoculate Vero bottles. On day 0, Vero cell confluence bottles were counted to calculate the virus PFU required to infect the bottle at 0.001 MOI. These bottles were infected with 1 mL of DENVax-4, DENVax-4b-P1, DENVax-4b-P10, or DENVax-2 or other composite constructs (eg, DENVax-4e, 4h, etc.). The virus was adsorbed onto the monolayer by shaking every 8 minutes for 90 minutes. After adsorption, 10 mL of cDMEM without FBS supplemented with 1% F-127 was added to each bottle, and then the samples were incubated at 37 ° C and 5% CO ₂ . Samples were collected from each bottle of supernatant on Day 2 and Days 4-12. All the supernatants in the collection bottles were collected on day 4 and on days 6-12, and the vaccine was replaced with the newly prepared cDMEM-F127. During the medium change, the cells were washed 3 times with PBS. The samples were stabilized in 1x FTA (FTA: 15% trehalose, 1% F-127, 0.1% human serum albumin, PBS), and then plaque titration was performed in the manner described previously to determine the titer.

After DENVax-4 and DENVax-4b were blindly passaged 10 times in Vero cells, each passage was sequenced to identify mutations between P1 and P10. Sequencing reactions of DENVax-4-P2-P1 and P10 and DENVax-4b-P3-P1 and P10 were performed in CDC. Viral RNA was isolated from viral stocks using the QIAmp viral RNA kit. Reverse transcriptase PCR (RT-PCR) was used to transcribe RNA into DNA using primers previously designed by CDC. The primers were designed from the sequences of DENV-2 16681 and DENV-4 1036. Using RT-PCR, approximately 7-9 PCR fragments were amplified from each construct. Then use Beckman Coulter to determine the sequence of the DNA fragments using an automatic sequencing reaction, and then align them for alignment.

    Sequencing    

After the various constructs have been passaged 10 or more times in Vero cells, each passage is sequenced to identify mutations. Sequencing reactions were performed on each sample, and viral RNA was isolated from virus stocks using the QIAmp viral RNA kit. Reverse transcriptase PCR (RT-PCR) was used to transcribe RNA into DNA using primers previously designed by CDC. The primers were designed from the sequences of DENV-2 16681 and DENV-4 1036. Using RT-PCR, approximately 7-9 PCR fragments were amplified from each construct. Then use Beckman Coulter to determine the sequence of the DNA fragments using an automatic sequencing reaction, and then align them for alignment.

    Research on non-human primates    

Cynomolgus monkeys were placed in different study groups and vaccinated with a tetravalent DENVax dose with various DEN-4 constructs. Each group tested one formula. Formulation 1 may contain high doses of DENVax with DENVax-4 1st generation, while primates in cohort 1 were given the first two doses on day 0 without additional. Formulation 2 contained a high dose of DENVax with a DEN-4 construct, and the primates in cohort 2 were given the first dose of 2 doses on day 0 without addition. Vaccination can be done subcutaneously or ID, or by other methods, using needles or needleless systems and syringes. Samples for serological analysis can be taken on days 0, 28, 58, 73, 90, 128 or other suitable time points. Neutralizing antibody response in serum was measured using plaque reduction neutralization technology.

    Plaque Reduction Neutralization Technique    

To test the neutralizing antibody yield in serum samples, a plaque reduction neutralization assay can be used. Two days prior to vaccination, inoculate Vero 6-well culture dishes to ensure single-layer confluence. Sequentially dilute serum samples twice in BA-1 dilutions in 96-well culture dishes, and then incubate dengue virus at 4 ° C for approximately 20 hours. After incubation, Vero cells were seeded with the prepared virus / serum dilution. The sample was allowed to adsorb for 90 minutes under vibration every 8 minutes to prevent the monolayer from drying. After adsorption, the wells were covered with 1: 1 BSS and agar solution, and then incubated at 37 ° C for 4 days. On day 4, cells were covered with 1: 1 ratio of BSS and agar supplemented with neutral red solution. Plaques were visible on the wells on days 5, 6, and 7. The GMT value means the average dilution of serum that neutralizes 50% of the virus. This is measured by determining the number of plaques formed in the absence of serum, divided by 2 (considering dilution), and then paying attention to which diluted serum causes plaque formation equal to or less than that number.

Compared with DENVax-4e (capsid mutation), the higher immunogenicity of DENVax-4h (envelope mutation) suggests that DENVax-4 envelope protein may be optimized. Envelope proteins provide epitopes for the production of neutralizing antibodies, and modification of sequences that optimize epitopes that elicit a strong antibody response can increase antibody titer. The mantle mutation at position 417 of DENVax-4h is a conservative part of the stem area. Compared to other flaviviruses, DENV-4 has a different amino acid at this position. The stem region is located in domain III of the E protein, and the strongest neutralizing epitope is located here. Binding and neutralizing the antibody at this position will prevent the stem region from fusing with the endosome membrane after endocytosis. In fact, in the flavivirus family including DENV-1, -2, -3, and WNV, position 417 is conservative. Reverting from E to K can change the substructure for a more robust immune response. As can be seen in other flaviviruses, further mutations can revert to conservatively charged aspartic acid (D).

The preceding discussion of the present invention is for the purpose of illustration. The foregoing is not intended to limit the invention to the form disclosed herein. Although the description of the present invention includes one or more specific examples and certain changes and modifications, other changes and modifications fall within the scope of the present invention. For example, those skilled in the art can understand the technology after understanding this disclosure. As well as understanding. This invention intends to obtain a structure, function, scope, or step that includes optional specific examples to such claimed ranges, including replacement, interchangeability, and / or equivalent, regardless of such replacement, interchangeability, and / or equivalent structure, Whether the function, scope or step is disclosed here, and any patentable subject matter is not intended to be disclosed publicly.

<110> Takeda Vaccine Co., Ltd. U.S. Government, represented by Minister of Health and Human Services

<120> Composition, method, and use of a dengue virus serotype 4 construct

<130> PI-56921-D1-GE

<150> 61 / 724,190

<151> 2012-11-08

<150> 61 / 788,536

<151> 2013-03-15

<160> 27

<170> PatentIn version 3.5

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Claims (25)

    A nucleic acid molecule comprising a chimeric flavivirus construct comprising a nucleic acid sequence encoding a non-structural protein and one or more structural proteins from a live, attenuated dengue-2 virus, and at least A nucleic acid sequence encoding one or more structural proteins from a second flavivirus selected from dengue-1, dengue-3, or dengue-4 virus, wherein the chimeric construct further comprises one or more Mutations, which include: mutations in the envelope (E) protein, when the E protein is from the second flavivirus, the mutation is at a position synonymous with the amino acid position 417 of dengue-4 Where the mutation in the E protein at the position synonymous with amino acid position 417 changes the amino acid to a positively charged residue; the mutation in the capsid (C) protein, the mutation is in the The live attenuated dengue-2 virus has an amino acid position 107 synonymous with the amino acid position, wherein the mutation in the protein C at the amino acid position synonymous with the amino acid position 107 is in the living Changed to an aromatic amino acid residue in attenuated dengue-2; And a mutation in NS4A at the amino acid position synonymous with amino acid position 17 of the live attenuated dengue-2 virus.         The nucleic acid molecule of claim 1, wherein the second flavivirus is dengue-4.         For example, the nucleic acid molecule of claim 2, wherein the amino acid positions 102-106 of attenuated dengue-2 are replaced by the synonymous amino acids of dengue-4 virus at these positions.         The nucleic acid molecule of claim 2, wherein the dengue-4 line is derived from strain 1036.         The nucleic acid molecule according to claim 1, wherein the position synonymous with 417 is an lysine rather than a lysine in the second flavivirus.         The nucleic acid molecule of claim 1, wherein the aromatic amino acid at the position synonymous with amino acid 107 is tyrosine.         The nucleic acid molecule of claim 1, wherein the mutation at the position in the NS4A protein synonymous with amino acid 17 changes the methionine to a basic amino acid.         The nucleic acid molecule of claim 1, wherein the mutation at the position in the NS4A protein synonymous with amino acid 17 changes the methionine to leucine.         The nucleic acid molecule of claim 1, wherein the live attenuated dengue-2 contains a mutation at position 57 in 5'NCR, a mutation at position 53 of NS1, and a mutation at position 250 of NS3.         The nucleic acid molecule of claim 1, wherein the live attenuated dengue-2 contains a mutation at position 53 of NS1 and a mutation at position 250 of NS3.         The nucleic acid molecule of claim 1, further comprising a structural protein from a third different flavivirus.         The nucleic acid molecule of claim 11, wherein the third different flavivirus is selected from the group consisting of West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne brain Inflammation virus and yellow fever virus.         The nucleic acid molecule of claim 1, wherein the live attenuated dengue-2 virus contains a mutated dengue-2 PDK-53 backbone.         A composition comprising one or more nucleic acid molecules as claimed in claim 1 and a pharmaceutically acceptable carrier.         The composition of claim 14, wherein the composition further comprises an additional dengue-dengue chimera.         The composition of claim 14, wherein the composition is part of a bivalent, trivalent, or tetravalent dengue virus composition.         The composition of claim 14, wherein the composition further comprises other live attenuated flaviviruses.         A composition comprising one or more polypeptides encoded by one or more nucleic acid molecules as claimed in claim 1 and a pharmaceutically acceptable carrier.         A use of a composition as claimed in claim 14 for the manufacture of a medicament for inducing an immune response to dengue virus in an individual.         The use of claim 19, wherein an immune response to at least one of dengue-1, dengue-2, dengue-3, and dengue-4 is induced in the individual.         A kit comprising one or more compositions as claimed in claim 14 and a container.         A live attenuated virus comprising a nucleic acid molecule as claimed in claim 1.         A live attenuated dengue-4 chimera, comprising a polynucleotide sequence represented by a sequence identification number: 22 (DENVax4e), a sequence identification number: 9 (DENVax4i), or a sequence identification number: 24 (DENVax4h).         A composition comprising one or more live attenuated dengue-4 chimeras as claimed in claim 23, and a pharmaceutically acceptable carrier.         A use of a composition as claimed in claim 24 in the manufacture of a medicament for inducing an immune response to dengue virus in an individual.